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A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

Azouaoui H, Montigny C, Ash MR, Fijalkowski F, Jacquot A, Grønberg C, López-Marqués RL, Palmgren MG, Garrigos M, le Maire M, Decottignies P, Gourdon P, Nissen P, Champeil P, Lenoir G - PLoS ONE (2014)

Bottom Line: Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS.We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation.This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, UMR 8221, Orsay, France; CEA, iBiTec-S (Institut de Biologie et de Technologies de Saclay), SB2SM (Service de Bioénergétique, Biologie Structurale et Mécanismes), Laboratoire des Protéines Membranaires, Gif-sur-Yvette, France; CNRS, UMR 8221, Gif-sur-Yvette, France.

ABSTRACT
P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼ 1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1:1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

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Streptavidin-based purification of Drs2p and Cdc50p.N-terminally Bad-tagged Drs2p and His10-tagged Cdc50p were purified onto a streptavidin column, and eluted by TEV cleavage. (A) Coomassie Blue stained 10% SDS-PAGE. (B, C, D) Immunodetection using a Biotin probe, a α-Drs2p antibody and a Histidine probe, as indicated. T, total membranes before solubilization; S, DDM-solubilized fraction; FT, flow-through; R, Streptavidin Sepharose resin before addition of TEV; RTEV, Streptavidin Sepharose resin after incubation with TEV for 16 h at 6°C; E, fraction eluted from the resin after incubation with TEV. Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated form of Cdc50p; His6-TEV, N-terminally hexahistidine-tagged TEV.
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pone-0112176-g002: Streptavidin-based purification of Drs2p and Cdc50p.N-terminally Bad-tagged Drs2p and His10-tagged Cdc50p were purified onto a streptavidin column, and eluted by TEV cleavage. (A) Coomassie Blue stained 10% SDS-PAGE. (B, C, D) Immunodetection using a Biotin probe, a α-Drs2p antibody and a Histidine probe, as indicated. T, total membranes before solubilization; S, DDM-solubilized fraction; FT, flow-through; R, Streptavidin Sepharose resin before addition of TEV; RTEV, Streptavidin Sepharose resin after incubation with TEV for 16 h at 6°C; E, fraction eluted from the resin after incubation with TEV. Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated form of Cdc50p; His6-TEV, N-terminally hexahistidine-tagged TEV.

Mentions: In previous experiments performed with solubilized crude membranes, we found that for protection of Drs2p from detergent-induced irreversible inactivation, DDM at a moderate concentration was one of the less deleterious detergents. DDM, for instance, was superior to C12E8 or Triton X-100 [36], and our purification procedure was therefore developed in the presence of DDM. Purification of the Drs2p-Cdc50p complex was achieved thanks to the strong interaction between the biotinylated Bad tag of Drs2p and an avidin-based resin. Figure 2 shows typical data obtained from N-terminally tagged constructs.


A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

Azouaoui H, Montigny C, Ash MR, Fijalkowski F, Jacquot A, Grønberg C, López-Marqués RL, Palmgren MG, Garrigos M, le Maire M, Decottignies P, Gourdon P, Nissen P, Champeil P, Lenoir G - PLoS ONE (2014)

Streptavidin-based purification of Drs2p and Cdc50p.N-terminally Bad-tagged Drs2p and His10-tagged Cdc50p were purified onto a streptavidin column, and eluted by TEV cleavage. (A) Coomassie Blue stained 10% SDS-PAGE. (B, C, D) Immunodetection using a Biotin probe, a α-Drs2p antibody and a Histidine probe, as indicated. T, total membranes before solubilization; S, DDM-solubilized fraction; FT, flow-through; R, Streptavidin Sepharose resin before addition of TEV; RTEV, Streptavidin Sepharose resin after incubation with TEV for 16 h at 6°C; E, fraction eluted from the resin after incubation with TEV. Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated form of Cdc50p; His6-TEV, N-terminally hexahistidine-tagged TEV.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230938&req=5

pone-0112176-g002: Streptavidin-based purification of Drs2p and Cdc50p.N-terminally Bad-tagged Drs2p and His10-tagged Cdc50p were purified onto a streptavidin column, and eluted by TEV cleavage. (A) Coomassie Blue stained 10% SDS-PAGE. (B, C, D) Immunodetection using a Biotin probe, a α-Drs2p antibody and a Histidine probe, as indicated. T, total membranes before solubilization; S, DDM-solubilized fraction; FT, flow-through; R, Streptavidin Sepharose resin before addition of TEV; RTEV, Streptavidin Sepharose resin after incubation with TEV for 16 h at 6°C; E, fraction eluted from the resin after incubation with TEV. Drs2p(t), truncated form of Drs2p; Cdc50p(g), glycosylated form of Cdc50p; His6-TEV, N-terminally hexahistidine-tagged TEV.
Mentions: In previous experiments performed with solubilized crude membranes, we found that for protection of Drs2p from detergent-induced irreversible inactivation, DDM at a moderate concentration was one of the less deleterious detergents. DDM, for instance, was superior to C12E8 or Triton X-100 [36], and our purification procedure was therefore developed in the presence of DDM. Purification of the Drs2p-Cdc50p complex was achieved thanks to the strong interaction between the biotinylated Bad tag of Drs2p and an avidin-based resin. Figure 2 shows typical data obtained from N-terminally tagged constructs.

Bottom Line: Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS.We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation.This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

View Article: PubMed Central - PubMed

Affiliation: Univ Paris-Sud, UMR 8221, Orsay, France; CEA, iBiTec-S (Institut de Biologie et de Technologies de Saclay), SB2SM (Service de Bioénergétique, Biologie Structurale et Mécanismes), Laboratoire des Protéines Membranaires, Gif-sur-Yvette, France; CNRS, UMR 8221, Gif-sur-Yvette, France.

ABSTRACT
P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼ 1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover a fraction that mainly contained a 1:1 complex, which was assessed by size-exclusion chromatography and mass spectrometry. The functional properties of the purified complex were examined, including the dependence of its catalytic cycle on specific lipids. The dephosphorylation rate was stimulated in the simultaneous presence of the transported substrate, phosphatidylserine (PS), and the regulatory lipid phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide that plays critical roles in membrane trafficking events from the trans-Golgi network (TGN). Likewise, overall ATP hydrolysis by the complex was critically dependent on the simultaneous presence of PI4P and PS. We also identified a prominent role for PI4P in stabilization of the Drs2p-Cdc50p complex towards temperature- or C12E8-induced irreversible inactivation. These results indicate that the Drs2p-Cdc50p complex remains functional after affinity purification and that PI4P as a cofactor tightly controls its stability and catalytic activity. This work offers appealing perspectives for detailed structural and functional characterization of the Drs2p-Cdc50p lipid transport mechanism.

Show MeSH
Related in: MedlinePlus